Persistent and enhanced expression of c-fos gene products in various kinds of human hematopoietic cell lines. Immunofluorescence study using prepared monoclonal antibodies

Two kinds of monoclonal antibodies recognizing fos proto-oncogene (c-fos) products were prepared using a synthetic oligopeptide corresponding to amino acids 127-152 of the fos oncogene products. These monoclonal antibodies (FO-120 & FO-145) detected fos gene products induced in a human monocyte cell line (U-937) by phorbol acetate (TPA) and induced in both human and mouse fibroblast cell lines (284, BALB/c 3T3) by serum-stimulation. One of the monoclonal antibodies (FO-120) reacted with 50-kDa and 42-kDa proteins and the other antibody (FO-145) reacted with a 30-33-kDa protein. The expression of the fos gene in various human hematopoietic cell lines was investigated using these prepared monoclonal antibodies. While almost all hematopoietic cell lines tested reacted with these monoclonal antibodies to various degrees, the majority of normal peripheral blood lymphocytes cultured with lectin (PHA) and interleukin 2 (IL-2) did not, suggesting that cells of some permanent hematopoietic cell lines, irrespective of their lineage specificity and growth factor dependency, continuously express the fos oncogene. These monoclonal antibodies may be useful for detecting early neoplastic changes in hematopoietic cells.     

Antigenic mimicry of a human cellular polypeptide by Mycoplasma hyorhinis.

A 46-kilodalton (kDa) polypeptide was immunoprecipitated from radiolabeled extracts of human cell lines infected with Mycoplasma hyorhinis by murine monoclonal antibodies PF/2A and ML77. Both of these antibodies also reacted in an enzyme-linked immunosorbent assay (ELISA) with M. hyorhinis cells and with human and nonhuman cell lines infected with M. hyorhinis but failed to react with A7573 cells infected with any of 10 other species of the order Mycoplasmatales. PF/2A also reacted in the ELISA with certain human cell lines that were demonstrated to be free of mycoplasma infection. From extracts of these lines, a polypeptide antigen that appeared as a 24-kDa doublet on polyacrylamide gels was immunoprecipitated by PF/2A. When the PF/2A-reactive human cell lines were infected by M. hyorhinis, both the 46- and 24-kDa antigens were immunoprecipitated by PF/2A. ML77 did not react in the ELISA with any noninfected human cells tested and failed to immunoprecipitate a 24-kDa component from any human cells. In Western blotting analyses of extracts of M. hyorhinis cells, both PF/2A and ML77 stained a 46-kDa band. PF/2A also stained 24-kDa bands in Western blotting analyses of reactive human cells and M. hyorhinis cells, although a 24-kDa component was not precipitated from extracts of M. hyorhinis cells by PF/2A.     

The isolation of hybrid cell lines producing monoclonal antibodies against the p15(E) protein of ecotropic murine leukemia viruses.

Hybrid cell lines were prepared by the fusion of mouse myeloma cells with the spleen cells of C57BL/6 mice that were immunized with the AKR leukemia K36. Approximately 10% of the hybrid cells produced immunoglobulins that reacted in antibody binding assays with AKR MuLV. By the combined use of low-density passage and cloning, seven independent cell lines were isolated. These cells produced antiviral antibodies at a level of 3–15 μg/ml of culture fluid. Inoculation of the hybrid cells into syngeneic mice resulted in the formation of tumors (hybridomas) that secreted extremely high levels of monoclonal antibodies (5–15 mg/ml) into the serum or ascites fluid. Five of the hybrid cell lines produced immunoglobulins of the IgM subclass, one produced IgG_2a, and one produced IgG_2b. In high-resolution two-dimensional polyacrylamide gels, these immunoglobulins showed the limited heterogeneity in heavy and light chains that would be expected for monoclonal products. Radioimmune precipitation assays demonstrated that the monoclonal antiviral antibodies reacted with the p15(E) protein of ecotropic MuLV; these antibodies did not react with the p15(E) protein of xenotropic MuLV. In contrast, rabbit antiserum prepared against purified p15(E) reacted equally well with ecotropic and xenotropic MuLV. The sera or ascites fluids from hybridoma-bearing mice had antibody titers 75- to 100-fold higher than the sera from conventionally immunized mice or rabbits. Serological analysis demonstrated that the monoclonal antibodies reacted with the cell surface of virus-producing leukemia cells, but not with normal thymocytes. Furthermore, monoclonal anti-pl5(E) antibodies of the IgG_2a subclass mediated lysis of the virion of ecotropic MuLV in the presence of complement.     

Shared antigens of human prostate cancer cell lines as defined by monoclonal antibodies

Eight monoclonal antibodies (MAbs) raised against human prostate cancer cell lines are described. One MAb was derived from the fusion of mouse myeloma P3x63Ag8-653 cells with spleen cells of mice immunized with DU145 prostate cancer cells. The other seven were from the fusion of myeloma lines P3x63Ag8-653 or SP2/0 with spleen cells of mice immunized with PC3, DU145 and 1013L prostate cancer cells. All of the antibodies also reacted with cell lines of other human cancer types, especially carcinomas. Immunoperoxidase staining on fixed tissue revealed strong reactivity only with antibody PrN10. Seven other antibodies seemed to bind to cell surface-associated (glyco)proteins. Antibodies PrL22 and PrO11 showed similar reactivity in radioimmunoassay, and immunoprecipitated a 160 kD molecular weight polypeptide from [125I]lactoperoxidase-labeled cells. Antibodies PrHk an PrQ12 bound to molecules with apparent MW of 115 kD and 100 kD, respectively; antibodies PrM24 and PrP14 revealed a more complex picture in immunoprecipitation of surface-labeled cells.     

Monoclonal Antibodies to Human Choriocarcinoma

ABSTRACT: We have established two monoclonal antibodies (TM7-3 and TM3-8) that react to choriocarcinoma cells. Both of these monoclonal antibodies have shown a similar reactive pattern to human cell lines, normal and neoplastic trophoblast tissues, and other fetal and adult tissues. They have reacted to nine of the ten choriocarcinoma cell lines, as well as to Hela cells (a cervical carcinoma cell line). During a cellular radioimmunoassay, neither TM7-3 nor TM3-8 reacted to two T lymphoblastoid cell lines or three B lymphoblastoid cell lines. Immunofluorescence and immunoper-oxidase staining showed that both monoclonal antibodies reacted selectively to the cytotrophoblast-like tumor cells of a choriocarcinoma and a hydatidiform mole but not to syncytiotrophoblast-like tumor cells. TM7-3 and TM3-8 also reacted slightly to the normal cytotrophoblast of early human chorionic villi under the same conditions as they did to choriocarcinoma tissue, but not to syncytiotrophoblast. In various normal tissues, TM7-3 and TM3-8 bind only to a part of the urinary tubles of the kidney and to the ducts of the pancreas of both adult and fetus.     

Detection of Rickettsia prowazekii in body lice and their feces by using monoclonal antibodies

In order to identify Rickettsia prowazekii in lice, we developed a panel of 29 representative monoclonal antibodies selected from 187 positive hybridomas made by fusing splenocytes of immunized mice with SP2/0-Ag14 myeloma cells. Immunoblotting revealed that 15 monoclonal antibodies reacted with the lipopolysaccharide-like (LPS-L) antigen and 14 reacted with the epitopes of a 120-kDa protein. Only typhus group rickettsiae reacted with the monoclonal antibodies against LPS-L. R. felis, a recently identified rickettsial species, did not react with these monoclonal antibodies, confirming that it is not antigenically related to the typhus group. Monoclonal antibodies against the 120-kDa protein were highly specific for R. prowazekii. We successfully applied a selected monoclonal antibody against the 120-kDa protein to detect by immunofluorescence assay R. prowazekii in smears from 56 wild and laboratory lice, as well as in 10 samples of louse feces infected or not infected with the organism. We have developed a simple, practical, and specific diagnostic assay for clinical specimens and large-scale epidemiological surveys with a sensitivity of 91%. These monoclonal antibodies could be added to the rickettsial diagnostic panel and be used to differentiate R. prowazekii from other rickettsial species.     

Western immunoblotting analysis of the antibody responses of patients with human monocytotropic ehrlichiosis to different strains of Ehrlichia chaffeensis and Ehrlichia canis.

In order to evaluate the relative sensitivity of the detection of antibodies against various antigenic proteins of Ehrlichia chaffeensis for the diagnosis of the emerging infectious disease human monocytotropic ehrlichiosis, Western immunoblotting was performed with 27 serum samples from convalescent patients with antibodies, as demonstrated by indirect immunofluorescence assay. Among 22 patients with antibodies reactive with the 120-kDa protein, 15 showed reactivity with the 29/28-kDa protein(s) and the proteins in the 44- to 88-kDa range. Two of the serum samples with this pattern reacted with the 29/28-kDa protein(s) of only the 91HE17 strain, and one sample reacted with only that of the Arkansas strain, indicating that the antibodies were stimulated by strain-specific epitopes. Overall, antibodies to the 29/28-kDa protein(s) were detected in only 16 patients' sera, suggesting that this protein is less sensitive than the 120-kDa protein. Two of 12 serum samples from healthy blood donors had antibodies reactive with the 120-kDa protein; one of these samples reacted also with the 29/28-kDa protein(s) of Ehrlichia canis, suggesting that unrecognized ehrlichial infection might have occurred, including human infection with E. canis. A high correlation between reactivity with the 120-kDa protein by Western immunoblotting and the recombinant 120-kDa protein by dot blot supports the potential usefulness of this recombinant antigen in diagnostic serology.     

Monoclonal antibodies to sheep MHC class II molecules recognize all HLA-D or subsets of HLA-D region products

Six murine monoclonal antibodies raised against sheep MHC class II molecules were analyzed for reactivity with HLA-D subregion products. All the antibodies reacted with human peripheral blood lymphocytes, monocytes, and B-lymphoblastoid cell lines homozygous for various HLA-DR specificities, suggesting that the antibodies recognized nonpolymorphic determinants on HLA class II molecules. SDS-PAGE and two-dimensional NEPHGE/SDS-PAGE analyses of molecules immunoprecipitated from 35S-methionine-labeled, DR-homozygous B-lymphoblastoid cell lines showed that the monoclonal antibodies precipitated typical class II molecules (Mr 32-34K and 25-29K). From comparison with antibodies of known HLA-D subregion specificity, two of the sheep antibodies appeared to react with the products of single HLA-D subregions, while another showed balanced reactivity with all HLA-D molecules. Antibody SBU.II 38-27 reacted exclusively with HLA-DQ molecules, antibody SBU.II 28-1 with HLA-DP molecules, and antibody SBU.II 49-1 with HLA-DR, -DQ, and -DP molecules. However, analysis of immunoprecipitates from surface-iodinated WT-49 cells (DR3 homozygous) using SBU.II 28-1 and the DP-specific monoclonal antibody B7/21, suggested that the two antibodies immunoprecipitated different alpha polypeptides. The two antibodies SBU.II 38-27 and 28-1 appear to be at least as specific as existing reagents, if not more so. As such, they are of value in their potential contribution to our understanding of the molecular characteristics and ultimately the functions of the HLA-DQ and -DP subregion products, as well as the identification/characterization of HLA-D equivalents in other species.     

Analysis of human antibody epitopes on the 65-kilodalton protein of Mycobacterium leprae by using synthetic peptides.

In order to study antibody reactivity to the Mycobacterium leprae 65-kilodalton (kDa) antigen, peptides representing overlapping sequences of the 65-kDa protein were synthesized, and a recombinant protein expression system for r65-kDa was constructed. Mouse monoclonal antibodies and leprosy patient seroreactivity to peptides and r65-kDa were tested by an enzyme-linked immunosorbent assay. All seven of the monoclonal antibodies used in this study reacted with their previously defined epitopes when tested against peptides. All monoclonal antibodies also reacted with r65-kDa. Leprosy patient seroreactivity to peptides and r65-kDa was seen in about one-third of active multibacillary cases. Specimens from patients positive for antibodies to peptides were seen to recognize different epitopes than did mouse monoclonal antibodies used in this study. It is concluded that substantial differences exist between mouse monoclonal antibodies and human leprosy patient reactivity to the 65-kDa antigen and that human seroreactivity to the 65-kDa antigen is indicative of a highly elevated bacillary load.     

Three monoclonal antibodies defining distinct differentiation antigens associated with different high molecular weight polypeptides on the surface of human embryonal carcinoma cells

Two monoclonal antibodies (TRA-1-60 and TRA-1-81) recognizing distinct cell surface antigens on human embryonal carcinoma (EC) cells were produced and characterized. These antibodies reacted strongly with undifferentiated human EC cells in indirect radioimmunoassays (RIA) and immunofluorescence (IF) assays, but only weakly or not at all with cells derived from pluripotent EC cells differentiating in vitro or in xenograft tumors, nor with other germ cell tumor cell lines that did not also express the typical features of human EC cells. They did not react with murine teratocarcinoma cell lines. A survey of other human tumor cell lines and normal human tissues disclosed that molecules recognized by these antibodies are not confined to human EC cells but that cross-reacting epitopes appear on several neoplastic and normal tissues, although in a different anatomical pattern for each antibody. Both antibodies immunoprecipitated a major polypeptide (apparent molecular weight approximately 240,000) and a minor polypeptide (apparent molecular weight approximately 415,000) from lysates of 125I surface-labeled human EC cells, in this respect resembling another monoclonal antibody, 8-7D, previously described by Blaineau et al. (1,2) However, sequential immunoprecipitation revealed that each of the three antibodies reacted with different molecules of slightly different molecular weights. The epitopes defined by the present antibodies differ from those recognized by the other human EC cell-specific monoclonal antibodies that have been described and provide new markers for studying the differentiation of pluripotent human EC cells.     

Human monoclonal antibodies reactive with cell surface antigens on human leukemia cell lines: many antibodies are (auto)antibodies

Primary Epstein Barr Virus (EBV) transformants from peripheral blood mononuclear cells (PBM) established in macrocultures were screened for the secretion of antibodies reactive with cell surface antigens on one or another of two indicator human leukemic cell lines and fused with the HMMA2.11TG/O human fusion partner. Human monoclonal antibodies (HuMAbs) were readily obtained. Relative oligoclonality of the primary EBV macrocultures was documented by the number of antibody secreting hybridomas (1-100%). The method permitted preselection for fusion of transformants producing antibodies of certain specificities and/or class. Fourteen HuMAbs, primarily of the IgM class, have been obtained. Those IgM HuMAbs obtained from patients with active diseases, e.g. Acute Lymphoblastic Leukemia (3 HuMAbs), and HIV infection (4 HuMAbs), were found to have a relatively broad spectrum of reactivity with cell lines of various hematopoietic lineages and normal cells, although several show selective reactivity with T cell lineage tumors or a selected population of cells. HuMAbs from normal donors of both the IgG and IgM class were obtained. The IgM HuMAbs from one volunteer reacted primarily with autologous and allogeneic macrophages (autologous PBM from the other patients were not available) as well as a diverse number of hematopoietic cell lines. From others, the IgG HuMAbs demonstrated a more restricted spectrum of reactivity, while the IgM HuMAbs reacted with both autologous and allogeneic normal cells. Thus, the B cell repertoire contains cells capable of secreting cell surface reactive antibodies and many of these antibodies express characteristics of autoantibodies. Those that did not react with autologous or allogeneic PBM may react with other autoantigens which have been expressed on the malignant cells used as screening targets or may represent true antitumor antibodies.     

抗人脑神经元特异性烯醇化酶单克隆抗体的制备和鉴定

本文采用离子交换和凝胶过滤层析技术，分离纯化了人脑神经元特异性烯醇化酶，并以此为抗原免疫ＢＡＬＢ／ｃ小鼠，通过杂交瘤技术，获得了两株稳定分泌单克隆抗体的杂交瘤细胞株，命名为５Ｂ１和３Ａ８。两株杂交瘤细胞均分泌ＩｇＧ１型类抗体。间接ＥＬＩＳＡ实验显示：５Ｂ１与ＮＳＥ有特异性反应。与ＮＳＥ的同工酶－非神经元特异性烯醇化酶不发生交叉反应；３Ａ８与ＮＳＥ和ＮＮＥ有同等强度的阳性反应。免疫印迹实验结果表明：     

Serological characterization of human T-cell leukemia (lymphotropic) virus, type I (HTLV-I) small envelope protein

Murine monoclonal antibodies were developed against the protein products produced by murine C127 cells which had been transfected with a recombinant plasmid clone containing the human T-cell leukemia (lymphotropic) virus type I (HTLV-I) proviral DNA coding regions for part of env, px, and the 3' LTR. Four antibodies with different binding patterns were obtained. One of these antibodies, F1.6, reacted against HTLV-I infected cells but not against noninfected cell lines. This antibody also reacted with sucrose-gradient purified HTLV-I and -II particles with preferential binding against the HTLV-I preparation. The F1.6 antibody bound to two proteins of approximately 21 and 43 kDa in gradient purified HTLV-I preparations, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots, and to a 43-kDa protein in cell lysates of HTLV-I infected cell lines; the F1.6 antibody did not bind significantly to any HTLV-II proteins in western blots. The three other antibodies F1.1, F1.2, and F1.5, recognized the same size proteins, 21 and 43 kDa in the gradient purified viral preparations of HTLV-I and in the case of the F1.2 antibody, the same size proteins in purified virus preparation of HTLV-II and -III. The F1.2 and F1.5 antibodies bound not only to purified HTLV particles but also to a variety of cellular proteins in HTLV-I infected and noninfected cells suggesting that they recognized epitopes which were shared between HTLV proteins and normal cells. The identity of the 21-kDa viral protein is most likely that of the small envelope glycoprotein. The identity of the larger protein is undetermined.     

Identification and purification of novel internalin-related proteins in Listeria monocytogenes and Listeria ivanovii.

Monoclonal antibodies were generated against a 30-kDa protein fraction derived from culture supernatants of a Listeria monocytogenes strain complemented with additional copies of the prfA regulator gene. Several of the antibodies reacted specifically with a hitherto unidentified, secreted 30-kDa polypeptide. By immunoblot analysis, the expression of this 30kDa polypeptide was found to be dependent on the presence of the PrfA regulator protein. Microsequencing of peptides derived from the partially purified 30-kDa protein revealed homologies to the InlA and InlB polypeptides of L. monocytogenes, which are required for the internalization of the bacteria into nonphagocytic cell lines. This prompted us to term the 30-kDa polypeptide internalin-related protein (Irp). Irp-specific monoclonal antibodies cross-reacted with a 24-kDa polypeptide present in culture supernatants of Listeria ivanovii, indicating the existence of an Irp-related protein in this pathogenic Listeria species.     

Antigenic heterogeneity in high- and low-virulence strains of Rickettsia rickettsii revealed by monoclonal antibodies.

Previously it has been reported that strains of Rickettsia rickettsii that differ greatly in their ability to cause disease in guinea pigs are similar by serological and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses. In this study, we used monoclonal antibodies to the virulent R and the relatively avirulent HLP strains to investigate strain differences which might account for the disparate behavior of the strains in guinea pigs. Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of the R and HLP strains were nearly identical for polypeptides with apparent molecular weights greater than 32 kilodaltons (kDa). All of the monoclonal antibodies to a lipopolysaccharide-like antigen reacted equally well with antigen from both strains by immunoblotting. None of the antibodies to the lipopolysaccharide-like antigen protected mice against challenge with viable rickettsiae. Some antibodies reacted with both 120- and 155-kDa polypeptides of both strains in radioimmune precipitation and immunoblotting tests, and other antibodies reacted only with the homologous strain. The monoclonal antibodies cross-reacted with the heterologous strain in the enzyme-linked immunosorbent assay essentially either completely or not at all. The ability of the monoclonal antibodies to the 120- and 155-kDa polypeptides to protect mice against the two strains was correlated with the ability of the antibodies to react with the antigens in the enzyme-linked immunosorbent assay and radioimmune precipitation or immunoblotting tests. These results demonstrate that R and HLP antigens which appear identical in molecular weight differ in their compositions of antigenic determinants.     

Expression of the c-fos oncogene in chemically-induced mouse tumours and in human skin tumours.

Using a polyclonal antibody to fos oncoprotein and an immunofluorescent technique, we investigated expression of the fos oncogene in chemically-induced mouse tumours and human premalignant and malignant skin lesion. In the chemically induced tumours, the nuclei of almost all carcinoma cells stained uniformly with this antibody, while positive cells were observed in the outermost layers in the benign papillomas. In human tumours, a greater degree of nuclear staining was observed in cases of squamous cell carcinoma than in tissues from patients with Bowen's disease. Basal cell carcinoma and malignant melanoma with histological evidence of invasiveness of the tumour cells showed a higher expression of the fos gene product than that seen in histologically circumscribed tumour nests. Thus, a higher expression of the fos oncogene is closely related to the malignant progression of tumour cells, in particular, the extent of invasiveness.     

Murine hybridoma monoclonal antibodies against insulin: cross-reactivity with insulins of three species and blocking of insulin binding to its receptor

Six hybridoma cell lines secreting monoclonal antibodies against pig insulin and cross-reacting with human and bovine insulins were obtained. Five of these monoclonal antibodies were IgG1, kappa, one IgG2b, kappa; their pI values were in the range of pH 6.3-7.4 and dissociation constants of the insulin-antibody complexes were 0.3-2 X 10(-8) mol/l, as determined by an immunoradiometric inhibition assay. All of these antibodies reacted with sterically closely related determinants and blocked the binding of 125I-pig insulin to the receptor on human MOLT-4 cell line.     

Production of human monoclonal antibodies to myeloperoxidase.

Two mouse-human heterohybridomas secreting human antibodies to myeloperoxidase (MPO) were derived from the peripheral blood of a patient who developed microscopic polyarteritis as the result of long-term treatment with hydralazine. Forty-five immunoglobulin-secreting lines were obtained from the fusion of patient lymphocytes with the CB-F7 heteromyeloma cell line. Of these, two antibodies, one IgG and one IgM, bound to myeloperoxidase in solid phase ELISA and gave a perinuclear staining pattern on ethanol-fixed human neutrophil cytospin preparations. The staining patterns were similar to those seen with serum from the patient. Antigen-inhibition studies revealed that the affinity of the IgG monoclonal antibody was 28 times higher (k = 1.4 x 10(-7)) than the IgM antibody (k = 5 x 10(-5)). Cross-inhibition studies further suggested that the two monoclonal antibodies recognized the same epitope on MPO. Of the other secreting cell lines, none produced antibody which reacted with the panel of autoantigens used for testing. Neither mononuclear antibody reacted with this panel indicating that they were not simply polyreactive natural autoantibodies. These are the first human monoclonal antibodies to native myeloperoxidase to be reported.     

Preparation and characterization of monoclonal antibodies to enteric adenovirus types 40 and 41

Summary We have prepared monoclonal antibodies to each of the enteric adenoviruses types 40 and 41. Three different hybridoma cell lines were selected which produced antibody found to react by radioimmunoprecipitation with adenovirus (Ad) hexon antigens. One was specific for Ad 40, another for Ad 41, and a third one reacted with both types. When tested in an enzyme immunoassay against all 41 known human Ad types, the type-specific monoclonal antibody against Ad 40 reacted homotypically, as did the monoclonal antibody against Ad 41. In addition, these monoclonal antibodies neutralized the homologous enteric Ad type. The monoclonal antibody which reacted with both enteric Ad types also showed lower levels of reactivity with the group C adenoviruses types 2, 5, and 6. The monoclonal antibodies produced will provide a definitive means for rapid identification of specific Ad types, and will be useful in defining the relationship of enteric adenoviruses to other types.